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Genome-wide analysis of Staufen-associated mRNAs identifies secondary structures that confer target specificity.

Laver JD, Li X, Ancevicius K, Westwood JT, Smibert CA, Morris QD, Lipshitz HD - Nucleic Acids Res. (2013)

Bottom Line: We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos.First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts.These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8, Department of Cell & Systems Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6, Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6, Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8 and Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, Toronto, Ontario, Canada M5S 3E1.

ABSTRACT
Despite studies that have investigated the interactions of double-stranded RNA-binding proteins like Staufen with RNA in vitro, how they achieve target specificity in vivo remains uncertain. We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos. Analysis of the localization and functions of these transcripts revealed a number of potentially novel roles for Staufen. Using computational methods, we identified two sequence features that distinguish Staufen's target transcripts from non-targets. First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts. Second, the 3'UTRs of Staufen-bound transcripts are highly enriched for three types of secondary structures. These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs. Our results provide the first systematic genome-wide analysis showing how a double-stranded RNA-binding protein achieves target specificity.

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Schematic of the in silico assays for discovery of Staufen’s binding preferences. (A) Structural annotations used in the manuscript. (B) Examples of the 15 of 19 motif. Black represents a 15 of 19 motif, whereas light grey indicates partners of this motif. (C) Examples of the [19,15] structure. (D) Examples of the [19,15,4] structure. (E) Examples of the [19,15,4]&[12,10,2] SRS. (see ‘Materials and Methods’ and ‘Results’ sections for details).
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gkt702-F5: Schematic of the in silico assays for discovery of Staufen’s binding preferences. (A) Structural annotations used in the manuscript. (B) Examples of the 15 of 19 motif. Black represents a 15 of 19 motif, whereas light grey indicates partners of this motif. (C) Examples of the [19,15] structure. (D) Examples of the [19,15,4] structure. (E) Examples of the [19,15,4]&[12,10,2] SRS. (see ‘Materials and Methods’ and ‘Results’ sections for details).

Mentions: Throughout this article, in reference to a specific secondary structure: we use the term ‘paired’ to refer to RNA bases that participate in a canonical base pair (i.e. a Watson–Crick base pair or a G-U wobble); we use the term ‘mismatch’ to indicate two bases that are found across from one another in a secondary structure but are not canonical base pairs; we use the term ‘unpaired’ to refer to bases that do not have a corresponding partner base on the other strand of a stem. Mismatches only occur in internal loops and unpaired bases occur in either bulge loops or internal loops, although the latter need not contain any unpaired bases. For example, the right-hand internal loop indicated in the schematic in Figure 5A contains two mismatches and one unpaired base, whereas the bulge in Figure 5A contains three unpaired bases and no mismatches.


Genome-wide analysis of Staufen-associated mRNAs identifies secondary structures that confer target specificity.

Laver JD, Li X, Ancevicius K, Westwood JT, Smibert CA, Morris QD, Lipshitz HD - Nucleic Acids Res. (2013)

Schematic of the in silico assays for discovery of Staufen’s binding preferences. (A) Structural annotations used in the manuscript. (B) Examples of the 15 of 19 motif. Black represents a 15 of 19 motif, whereas light grey indicates partners of this motif. (C) Examples of the [19,15] structure. (D) Examples of the [19,15,4] structure. (E) Examples of the [19,15,4]&[12,10,2] SRS. (see ‘Materials and Methods’ and ‘Results’ sections for details).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814352&req=5

gkt702-F5: Schematic of the in silico assays for discovery of Staufen’s binding preferences. (A) Structural annotations used in the manuscript. (B) Examples of the 15 of 19 motif. Black represents a 15 of 19 motif, whereas light grey indicates partners of this motif. (C) Examples of the [19,15] structure. (D) Examples of the [19,15,4] structure. (E) Examples of the [19,15,4]&[12,10,2] SRS. (see ‘Materials and Methods’ and ‘Results’ sections for details).
Mentions: Throughout this article, in reference to a specific secondary structure: we use the term ‘paired’ to refer to RNA bases that participate in a canonical base pair (i.e. a Watson–Crick base pair or a G-U wobble); we use the term ‘mismatch’ to indicate two bases that are found across from one another in a secondary structure but are not canonical base pairs; we use the term ‘unpaired’ to refer to bases that do not have a corresponding partner base on the other strand of a stem. Mismatches only occur in internal loops and unpaired bases occur in either bulge loops or internal loops, although the latter need not contain any unpaired bases. For example, the right-hand internal loop indicated in the schematic in Figure 5A contains two mismatches and one unpaired base, whereas the bulge in Figure 5A contains three unpaired bases and no mismatches.

Bottom Line: We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos.First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts.These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8, Department of Cell & Systems Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6, Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6, Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8 and Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, Toronto, Ontario, Canada M5S 3E1.

ABSTRACT
Despite studies that have investigated the interactions of double-stranded RNA-binding proteins like Staufen with RNA in vitro, how they achieve target specificity in vivo remains uncertain. We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos. Analysis of the localization and functions of these transcripts revealed a number of potentially novel roles for Staufen. Using computational methods, we identified two sequence features that distinguish Staufen's target transcripts from non-targets. First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts. Second, the 3'UTRs of Staufen-bound transcripts are highly enriched for three types of secondary structures. These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs. Our results provide the first systematic genome-wide analysis showing how a double-stranded RNA-binding protein achieves target specificity.

Show MeSH